Insights into the structural and functional activities of forgotten Kinases: PCTAIREs CDKs

In cells, signal transduction heavily relies on the intricate regulation of protein kinases, which provide the fundamental framework for modulating most signaling pathways. Dysregulation of kinase activity has been implicated in numerous pathological conditions, particularly in cancer. The druggable nature of most kinases positions them into a focal point during the process of drug development. However, a significant challenge persists, as the role and biological function of nearly one third of human kinases remains largely unknown. Within this diverse landscape, cyclin-dependent kinases (CDKs) emerge as an intriguing molecular subgroup. In human, this kinase family encompasses 21 members, involved in several key biological processes. Remarkably, 13 of these CDKs belong to the category of understudied kinases, and only 5 having undergone broad investigation to date. This knowledge gap underscores the pressing need to delve into the study of these kinases, starting with a comprehensive review of the less-explored ones. Here, we will focus on the PCTAIRE subfamily of CDKs, which includes CDK16, CDK17, and CDK18, arguably among the most understudied CDKs members. To contextualize PCTAIREs within the spectrum of human pathophysiology, we conducted an exhaustive review of the existing literature and examined available databases. This approach resulted in an articulate depiction of these PCTAIREs, encompassing their expression patterns, 3D configurations, mechanisms of activation, and potential functions in normal tissues and in cancer. We propose that this effort offers the possibility of identifying promising areas of future research that extend from basic research to potential clinical and therapeutic applications. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-024-02043-6.


Supplementary Text, Related to figure 2 and paragraph 3 PCTAIRE 3D-Structure Analysis
Detailed structural and mechanistical studies on atypical CDKs are scarce.However, computational tools could aid in generating and then test specific hypothesis (4,5).Here, we used the computational tool developed by McSkimming.D et.al, to predict active/inactive state of kinases, based on their 3D-configurations (6).
We compared the CDK16 3D-structure with the ones of CDK1, CDK2 and CDK5, which are the closest homologues to PCTAIREs among CDKs for which experimental structure was available.
Corresponding PDB files were retrieved from Protein Data Bank database.
Obtained CDKs structures were annotated as either active or inactive, based on McSkimming D et al. (6).In total, we annotated 365 PDB files for CDK1, CDK2, CDK5 and CDK16 (either curated or predicted).CDK16 have two deposited structures in protein databank [PDB-ID: 5G6V_A and 3MTL_A].We used the CDK16 [PDB-ID: 5G6V_A] that, like the other available CDK structures used, displays the cyclin binding domain in a helical conformation.Most of the annotated structures belong to CDK2.For CDK1, we had two PDBs: in active [PDB-ID:4Y72_A] and inactive [PDB-ID: 4YC6_A] conformation.All available CDK5 structures available were in active configuration, either annotated or predicted.Annotated PDB files were used as input for multiple structure alignment with the local version of mTM-align software (7) with default settings at GARR (Italian National Computer Network for Research and Universities) high performance computer.Further, visual inspection and comparison of structures were performed with Chimera software (8), as illustrated in (Supplementary Figure 3a).Briefly, mTM software outputs two scoring functions: 1) TM-score (Template Scoring), a length-independent scoring function, which is a robust metric for overall structural similarity (score 1 means identical structures); 2) RMSD (Root Mean Square Deviation), a metric for local similarities in protein structures (lower RMSD reflects better local similarities between proteins).
Both TM and RMSD scores gave similar results among active and inactive structures and, based on these scores, CDK active and inactive structures clustered separately (Supplementary Figure 3b, c).To our surprise, the predicted inactive 3D-structure of CDK16 clustered with the active structures of CDK1, CDK2 and CDK5 (Supplementary Figure 3b, c).Visual inspection of conserved regions revealed a high degree of conservation at the core of CDK kinase domains (Supplementary Figure 3d; conserved regions, colored in blue, are mapped on CDK16 structure).
A deeper look into the specific motifs known to govern CDK activation (i.e., cyclin binding domain, DFG, HRD and CDK16 specific additional insertion in CDK/MAPK domain (9) (hereafter referred to as MAPK domain) revealed that only HRD is highly conserved among the analyzed CDKs.The residues flanking the cyclin binding, DFG and the MAPK motifs (but not themselves) are similarly well conserved (Supplementary Figure 3e).
Since hierarchical clustering revealed that inactive CDK16 structure is more similar to active than inactive CDK1, CDK2 and CDK5 structures, we looked for similarities/differences between CDK16 and the other CDKs in the motifs that undergo reconfiguration upon cyclin binding (i.e.cyclin binding domain, HRD, DFG).To this aim, we visually compared CDK16 to CDK1, CDK2 and CDK5 structures, as detailed below.
First, heatmap analyses confirmed that CDK16 had relatively higher TM and lower RMSD scores with active CDK1 and CDK2 structures, compared to inactive ones (Supplementary Figure 4a, b).
Pairwise comparison of CDK16 with active and inactive CDK2 confirmed that it has relatively high global similarity with active CDK2 configuration (Supplementary Figure 4c).The cyclin binding motif of CDK16 (PCTAIRE) is slightly more similar to active CDK2 cyclin binding motif (PSTAIRE), compared to the inactive one (Supplementary Figure 4c, inset a).The DFG motif of CDK16 does not overlap with the corresponding motif of CDK2 structure, while the topology of the HRD motif is quite similar comparing CDK16, with both active and inactive CDK2 structure.
Similarly, the active CDK1 structure [PDB-ID: 4Y72] shares the lowest RMSD score with CDK16 (Supplementary Figure 4d) and, accordingly, CDK16 displays a better overlap with active than with inactive CDK1 3D-configuration (Supplementary Figure 4d).Moreover, the PCTAIRE motif shows better spatial overlap with the PSTAIRE motif of active than inactive CDK1 (Supplementary Figure 4d, inset 1).Differently from CDK2, the active CDK1 cyclin binding domain overlaps well with the corresponding domain of CDK16 (compare Supplementary Figure 4c inset1 with 4d inset1).Finally, while the DFG motif of CDK16 is topologically different from that of CDK1 (Supplementary Figure 4d, inset 2), the HRD motifs of CDK16 and active/inactive CDK1 structures are topologically highly similar to each other (Supplementary Figure 4d, inset 3).
Overall, these comparative analyses revealed a high degree of 3D similarity of inactive CDK16 with active CDK1 and CDK2 structures.
Next, we compared CDK16 to CDK5, the archetype of non-canonical CDK, observing a high degree of overall similarity between the 3D-structures of the two CDKs.Of note, all CDK5 structures annotated in PDB are deposited in active configuration and the cyclin-binding motifs of CDK16 and CDK5 have different 3D-coordinates (Supplementary Figure 5, inset 1).Additionally, DFG motif of CDK16 is spatially out of the coordinates of the corresponding CDK5 motif (Supplementary Figure 5, inset 2).Last, topology of HRD motif of CDK5 is similar to the one of CDK16 (Supplementary Figure 5, inset 3).Overall, these data suggested that the cyclin binding domains of CDK16 is topologically more similar to the corresponding domain of active CDK1.
Next, we compared CDK5 to CDK1 and CDK2 structures.TM and RMSD scores of CDK5 structures are relatively higher for active CDK1 and CDK2 structures compared to inactive ones (Supplementary Figure 6).We used CDK5 structures [PDB-IDs: 4AU8 and 1UNH], which have the highest TM (0.881) and the lowest RMSD (1.995), compared to CDK16.In general, CDK5 presents good similarity with active CDK1 and CDK2 structures (Supplementary Figure 6).CDK5 cyclin binding domain is spatially overlapping with active CDK2, but not with active CDK1 cyclin binding motif (Supplementary Figure 6, inset 1).DFG and HRD motifs of CDK5 are similar to both active CDK1 and CDK2 corresponding motifs (Supplementary Figure 6, inset 2 and 3, respectively).As mentioned, no curated or annotated CDK5 structures are categorized as inactive and, consequently, we were not able to distinguish configurational differences between active and inactive CDK5 structures with respect to CDK1 or CDK2.
Lastly, we evaluated whether the observed features, particularly the similarity in cyclin binding motif of CDK16 with active CDK1, are unique to CDK16 or shared among other PCTAIREs subfamily members.
Since CDK17 and CDK18 structures are yet to be experimentally determined, using the SWISS-Modell tool (10) we modelled CDK17 and CDK18 structures, using CDK16 structure as template [PDB-ID: 5G6V] and then visually compared them to CDK16.This approach revealed a high degree of shared geometry among the PCTAIRE family members (Supplementary Figure 7).We observed small differences at PCTAIRE and DFG motifs, between CDK18 in silico structures and the others (Supplementary Figure 7, inset 1-3).In particular, the side chain of phenylalanine of CDK18 DFG motif is notably different, compared to CDK16 and 17.This observation is in line with the structure-guided alignment results, which showed that similarity is comparatively higher between CDK16 and CDK17 (72.5 %) than between CDK16 and CDK18 (65.7 %) or CDK17 and CDK18 (69.7%) (see main text, Figure 1c and Supplementary Figure 2).
Overall, these analyses revealed that the structure of CDK16 in an inactive configuration is surprisingly more similar to the active than the inactive 3D-structures of CDK1, CDK2 and CDK5.
Of note, the cyclin binding domain of CDK16 overlaps very well with the corresponding motif of comprised among the conserved regions.Visualization was obtained with Chimera software (8).e) 2D view of CDK16 sequence and conserved regions, depicting PCTAIRE, HRD and DFG-spanning regions of CDK16 protein, visualized with Bioconductor package ggmsa.2a and   b).CDK5 shares overall high similarity with both active CDK1 and CDK2.More in detail, CDK5 cyclin binding motif overlaps better with the one of CDK2 than with the one of CDK1 (inset 1).Differently from CDK16, CDK5 DFG motif is on the same plane of both CDK1 and CDK2 DFG motifs (inset 2).The HRD motif of the 3 CDKs is also well topologically conserved (inset 3).

1 Supplementary Figure 5 .Supplementary Figure 6 .
PCTAIRE and DFG motifs are not conserved among CDK16 and CDK1, CDK2 or CDK5 structures, but their spanning residues are conserved.Different from PCTAIRE and DFG motifs, the 3Dcoordinates of HRD motif, as well as its spanning region, are conserved.Asterisks mark conserved residues.Supplementary Figure 4. Inactive CDK16 shares high similarity with active CDK1-2 structures, a) and b) Heatmap representation of TM and RMSD scores of CDK16 versus CDK1-2-5.c) CDK16 structure was superimposed with active CDK2 [2CTA_A] and inactive CDK2 [PDB-ID: 1OIQ_A] in Chimera.(8)Insets 1, 2, 3 are zoom-in view motifs.As shown, cycling binding motif of CDK16 has very close spatial coordinates to the respective motif of the active CDK1.DFG motif of CDK16 is out of the CDK1 and 2 DFG-motif planes.Superimposition of CDK16 structure with CDK5.CDK16 structure was superimposed with active CDK5 [PDB-ID:4AU8_A] and CDK5 [PDB-ID:1UNH_A] structures to comparatively visualize topological similarities/differences.Superimposition and visualization were performed as described earlier (see footnote Supplementary Figure4).Insets 1, 2, 3 are zoom-in views of PC(T)AIRE, DFG and HRD motifs, respectively.Pairwise comparison of CDK5 structures with CDK1 and 2. Superimposition of the indicated CDK5 structures with CDK1 and CDK2 was performed as described earlier (see footnote Supplementary